White Paper –Trends in the Manufacturing of Viral Gene Therapeutics and Next Generation Vaccines

Jeff Strobel, Site Director, SAFC Carlsbad, CA

Much of the technology used for the current manufacturing of viral vaccines has its roots in discoveries from the gene therapy industry. Gene therapy, or more correctly, gene transfer, is a catch-all phrase for those products which use a viral vector, plasmid, or cell as a delivery vehicle for a gene product. Early applications in this field really developed in the 1980s and 1990s, often using viral vectors such as adenovirus or retrovirus to deliver genes. There are still no approved gene therapy products in the western world, but numerous applications are being pursued, notably in cancer, central nervous system disorders and cardiac disease. There are also some exciting studies for diseases of the eye. In sum, researchers are now being more realistic than in the early days, seeing the future of gene therapy in providing effective treatments, not cures.

Viral Vectors and Virus Manufacturing for Gene Therapy and Vaccines

In the early days of gene therapy, the safety of viral vectors was a real issue, and two incidents in particular caused a great deal of concern. The first, a teenager being treated for ornithine transcarbamylase deficiency at the University of Pennsylvania, died four days after being treated with an adenovirus loaded with a corrected gene after a massive immune response to the virus. In the second incident, a group of French children with severe combined immunodeficiency, or ‘bubble boy’ disease, were cured but developed leukemia after the retrovirus vector integrated itself preferentially into certain sites. While still an experimental therapy, the dangers are now much better understood, and the issues facing companies with gene therapy products have a more defined standard of efficacy and safety.

In the early days of gene therapy, the safety of viral vectors was a real issue, and two incidents in particular caused a great deal of concern. The first, a teenager being treated for ornithine transcarbamylase deficiency at the University of Pennsylvania, died four days after being treated with an adenovirus loaded with a corrected gene after a massive immune response to the virus. In the second incident, a group of French children with severe combined immunodeficiency, or ‘bubble boy’ disease, were cured but developed leukemia after the retrovirus vector integrated itself preferentially into certain sites. While still an experimental therapy, the dangers are now much better understood, and the issues facing companies with gene therapy products have a more defined standard of efficacy and safety.

This increased confidence that gene therapy is not intrinsically unsafe means that several products are now in late stage trials. A few of note have been submitted for approval – Ark Therapeutics’ Cerepro® for glioma, and Introgen’s Advexin® for head and neck cancer – both have faced or are facing issues of overall efficacy. However, the maturing of the marketplace has resulted in a growing demand for larger quantities of viral product to be manufactured. This demand is being supplemented by the growth in importance of cell culture techniques for manufacturing vaccines.

There have been several drivers for the resurgence in vaccine research in the past. First, the lack of commercial funding led President Clinton to form the NIH Vaccine Research Center in 1999, with an initial goal of developing an HIV vaccine.

A second wave of interest came in the development of biodefense vaccines – many of which can be made via viral methods – in the aftermath of 9/11.

More recently, concerns over new flu viruses – first H5N1 bird flu and then H1N1 swine flu – have pushed demand for more efficient manufacturing methods and posed a significant test to operations and supply chains. Pandemics are, essentially, spikes in demand, and there has been an increasing use of CMOs to handle that spike. This also represents a business challenge for the CMOs themselves – there needs to be enough customers and projects in the core business that allows for the CMO to provide quality services, while also having the capabilities to handle surges in demand.

Cell Culture Virus Manufacture

When manufacturing viral products by mammalian cell culture, the two essential components are the cell line and the source virus. Historically, the two most important virus types were retrovirus and adenovirus, but in recent years many others have been introduced, both enveloped and non-enveloped, including lentivirus, adeno-associated virus, herpesvirus, alphavirus, coxsackie virus and reovirus. This gives users the ability to match the viral vector to the therapeutic or vaccine intent.

The cells for the culture can be grown in flat stock, where the cells adhere to a plastic surface, or in suspension. In either case, the aim is to grow the cells by feeding them the correct nutrients until they are ready for infection. Cells are increasingly being grown in suspension using serum-free media. The most common cell lines are HEK293, 293T and Crucell’s PER.C6® but, just as is happening in the monoclonal antibody field, there is a drive towards customized cell lines, such as those derived from Chinese hamster ovary cells. A recent development is the introduction of avian cell lines, with two examples – EB66® from Vivalis and ProBioGen’s AGE1.CR®. These are designed to be direct replacement for chicken embryo fibroblasts in vaccine manufacture and will likely have potential beyond this fairly narrow application.

Disposable Bioreactors

There is a growing trend towards the use of disposable equipment, with the goal of a closed system with one unit operation following seamlessly on from the next. At larger scales, say 50 L and above, clients still tend to be more comfortable with traditional stainless steel bioreactors, but the industry is definitely moving towards disposable bioreactors – even up to 1,000 L volumes.

At our Carlsbad facility, we have been using disposable reactors since we began manufacturing viruses in 1997. Containment is paramount in virus manufacture because of the universal precautions around infection risk, and using a closed, disposable bioreactor system adds a layer of containment. It also reduces change-over time between products. With traditional stainless steel bioreactors, cleaning validation is imperative because all traces of the first product must be removed before the second is made. If the bioreactors and associated equipment are disposable, there is very little chance that there will be any cross contamination.

While disposables introduce a higher variable cost, the overall cost is lower when all factors are taken into account. There is no need for the time-consuming cleaning and validation process, or the up-front investment in expensive steel vessels. It is also easier to account for the cost of running an individual product. If a facility is dedicated to one product, or only changes every few months, then stainless steel vessels remain the best option, but for a contract manufacturer who works on many different virus products, disposables make more sense.

This growing interest in using disposables has been supported by the entry of more suppliers into the market. Early pioneers, such as WAVE Biotech (now a part of GE Healthcare), have been joined by some of the big names in biomanufacturing such as Sartorius and Hyclone, and there are now multiple options to suit different applications.

The Importance of Containment

Containment is paramount when manufacturing viruses. Unlike a standard clean room facility, where air is pushed out of the room in a “clean to dirty” fashion, a containment facility works under negative air pressure. By pushing air into the room, it ensures that if there should be a spill or aerosolization of the virus, it will stay in the room and cannot escape and cause widespread contamination. In both our pilot facility and large-scale facility, 100% air inflow and completely separate exhaust significantly reduces the risk of cross-contamination. Our expansion space adds additional controls and capabilities, including continuous differential pressure monitoring, additional airlocks, a kill-tank, and a WFI.

The recent expansion of our Carlsbad facility has allowed us to provide larger client-specific areas. Our original pilot plant was designed for simultaneous multi-product manufacturing at scales up to 100 L. Facility design elements centered on personnel and material flows, air pressurization techniques, and efficient change-over. Our new space is designed for two, fully independent manufacturing trains and will be dedicated for campaigns and larger scale projects. Thus, projects that are at different scales and stages of the product lifecycle can be efficiently matched up with the appropriate clinical/commercial manufacturing space.